Highlighting Synaptic Communication in the Enteric Nervous System

Abstract

Light microscopic techniques date back to the end of the 17th century when Anthonie van Leeuwenhoek built an optical device with a series of self-ground lenses. These techniques were instrumental in discovering and elucidating the cellular organization and structure of many organisms. It took until the last decades, to develop fluorescent probes with specific biological or chemical function, before physiologic processes could be monitored. To this end, molecules were designed to either become membrane potential or ion (Ca2+, H+, …) sensitive, or to divulge the location of specific proteins. Apart from the synthetic molecules, genetically encoded protein dyes derived from jellyfish and corals have become extremely useful for labeling specific biological interactions or processes. The use of optical techniques to study cellular function has 2 important advantages. First, several cellular and subcellular structures can be studied simultaneously, which allows accurate detection of sequential events. Second, they always provide a spatial correlate to physiologic events of interest.The enteric nervous system (ENS), responsible for the coordination of many different gastrointestinal functions, consists of approximately 100 million neurons wired in a ganglionated network. Each of the neurons receives synaptic input from many different contacts (Figure 1), which adds layers of complexity to the network. The output of this nerve network controls motility by reflex activation of specific pathways leading to peristalsis. Alternatively, the ENS can be seen as a continuously oscillating network1Wood J.D. Enteric nervous system: reflexes, pattern generators and motility.Curr Opin Gastroenterol. 2008; 24: 149-158Crossref PubMed Scopus (79) Google Scholar that is tilted toward a different output, depending on subtle inputs from the periphery. It is well known that neural networks2Johnson B.R. Schneider L.R. Nadim F. et al.Dopamine modulation of phasing of activity in a rhythmic motor network: contribution of synaptic and intrinsic modulatory actions.J Neurophysiol. 2005; 94: 3101-3111Crossref PubMed Scopus (27) Google Scholar change their output depending on low concentrations of specific mediators. The unitary components that are modulated to produce the change may well be synapses and not neurons. In states of disease, such as inflammatory conditions, synaptic activity was shown to be altered dramatically and persistently.3Linden D.R. Sharkey K.A. Mawe G.M. Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon.J Physiol. 2003; 547: 589-601Crossref PubMed Scopus (162) Google Scholar, 4Lomax A.E. Mawe G.M. Sharkey K.A. Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.J Physiol. 2005; 564: 863-875Crossref PubMed Scopus (78) Google Scholar Therefore, to understand the output of the ENS, it is crucial to also appreciate the contributing synaptic signals, because these may be essential to determine output, although they do not always reach the “attention” level by generating an action potential in the postsynaptic cell. Electrode techniques5Wood J.D. Cellular neurophysiology of enteric neurons.in: Johnson L.R. Barret K.E. Ghisan F.K. Physiology of the gastrointestinal tract. 4th ed. Elsevier, San Diego2006: 629-664Crossref Scopus (6) Google Scholar and voltage-sensitive optical techniques6Schemann M. Michel K. Peters S. et al.Cutting-edge technology III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G919-G925Crossref PubMed Scopus (50) Google Scholar have generated invaluable information with high temporal resolution about fast and slow postsynaptic potentials in the ENS. In this review, we focus on how synaptic activity or neurotransmitter release itself, rather than the processed postsynaptic signal, can be measured optically.Imaging Synaptic EventsCytosolic Ca2+ MeasurementsCytosolic Ca2+ indicators are sensitive to changes in intracellular Ca2+ concentration, which reflect activity of the cell. They can be loaded (1–10 μmol/l, 20 minutes) in their acetoxymethyl (AM)-ester form, which renders the molecule lipophilic and allows it to leak through the cellular membrane. The dye becomes trapped inside the cell once cytosolic esterases have cleaved the AM-ester (Figure 2A). The quality of Ca2+ indicators (Fura-2, Fluo-3, Fluo-4) has improved immensely over the last decades and the signal to noise ratio of dyes like Fluo-4 makes it possible to easily measure signals from small cellular compartments, such as varicosities and synapses. Using this technique, spontaneous and evoked Ca2+ signaling events in fibers and varicosities can be recorded in cultured enteric neurons or in tissue. Using a custom developed computer algorithm (in Igor Pro, Wavemetrics, Lake Oswego, OR) that selects active pixels in an image sequence, we can construct so-called activity over time images that have the same quality of structural information as immunostaining, but in which a physiologic response is represented. Postprocessing of the sample with specific antibodies can further identify the characteristics of the fiber or the apposed postsynaptic cell. Fluo-4 signals have the advantage to be very bright, with a high signal to noise ratio; the loading procedure is in many ways random, but it is difficult, if not impossible, to load synaptic contacts embedded deeply in the tissue. Ca2+ imaging is easy to use and generates high-resolution information about release sites in the ENS, but one should bear in mind that a Fluo-4 signal just reflects the increase in cytosolic Ca2+ and is not conclusive proof for synaptic activity.Figure 2Imaging of synaptic activity in enteric nerve networks. (A) Ca2+ imaging in neuronal boutons or varicosities. (Top) Representative images of Fluo-4 loaded cultured myenteric neurons from guinea pig either at rest or while active. (Bottom left) Color-coded image representing Fluo-4 loading (green) and maximal “activity over time” (AoT) in red. (Bottom right) Graph showing the relative Fluo-4 fluorescence of 3 neuronal varicosities (color matched with arrows in top images) reflecting their spontaneous activity over time. Bars, 20 μm. (B) Synaptic vesicle recycling in cultured myenteric neurons. (Left) Image of enteric boutons labeled with FM1-43 before destaining protocol. *Neuronal cell bodies. (Right) Representative fluorescence traces of 2 enteric boutons (arrows, in left panel) destained by 3 consecutive stimuli (arrowhead and arrow, 40 and 400 single electrical stimuli; bar, 75 mmol/L K+ application). Bars, 20 μm. (C) SynaptopHluorin visualization in whole mount tissue. (Top) Images of synaptopHluorin in an enteric ganglion at pH 7.4 (left) and treated with NH4Cl (right), to alkalinize all intracellular compartments and reveal the presence of quenched synaptopHluorin molecules. (Bottom left) Immunolabeling of nitric oxide (NOS)–positive neurons, surrounded by synpatopHluorin expressing fibers, in a mouse myenteric ganglion. (Bottom right) Typical traces of single single-release events during which synaptopHluorin is unquenched and causes an increase in fluorescence. Bars, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Labeling Vesicle Recycling: FM1-43Because they were originally meant to become voltage-sensitive probes, FM dyes (FM1-43, FM2-10, FM-595) were designed to intercalate well and fluoresce mainly in a membrane lipid double layer. Because of this property, they became excellent tools to label membrane trafficking and vesicle turnover. The technique, pioneered by Betz and Bewick,7Betz W.J. Bewick G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.Science. 1992; 255: 200-203Crossref PubMed Scopus (648) Google Scholar has generated important information of how synapses organize their synaptic vesicles during neurotransmitter release. The principle is rather simple and involves 3 consecutive steps (Figure 3B). In a first step, FM dye is present in the extracellular medium and actively recycling release sites become labeled, because freshly endocytosed synaptic vesicles at the presynaptic membrane contain the dye. During a second step, the excess dye is washed away from the preparation and the active release sites remain fluorescent (Figure 2B). In a last step, boutons can be stimulated again so that they “destain,” the kinetics of which closely reflect the kinetics of vesicle release (Figure 2B).8Vanden Berghe P. Klingauf J. Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system.Neuroscience. 2007; 145: 88-99Crossref PubMed Scopus (18) Google Scholar With this dye, one can directly measure the dynamic properties of vesicle recycling and release in enteric neurons in primary culture as well as in dissected whole-mount tissue. Using this technique, we were able to show that enteric release sites have kinetic properties very similar to the central nervous system and that brain-derived neurotrophic factor was able to modulate release of enteric nerves caused by subtle electrical stimuli.9Boesmans W. Gomes P. Janssens J. et al.Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system.Gut. 2008; 57: 314-322Crossref PubMed Scopus (64) Google Scholar Owing to the specific loading procedure, FM dyes outperform Ca2+ dyes in selectivity to label synaptic contacts. However, stability of the FM dyes is worse and photobleaching is an important issue the user should consider. A decrease in fluorescence may compromise the “destain” kinetics, bleaching should be reduced as much as possible by keeping exposure times short.Figure 3Schematic of the principles and methods for visualizing synaptic vesicle release. (A) Simplified drawing showing the main characteristics of a presynaptic terminal. Upon arrival of an action potential, voltage gated Ca2+ channels open owing to depolarization and allow Ca2+ to flow into the terminal. This triggers the synaptic vesicles to fuse with the presynaptic membrane and release their neurotransmitter contents. (B) Schematic of 3 different optical techniques to visualize synaptic activity. (Left) The Ca2+ indicator Fluo-4 can be used to monitor the increase in intracellular Ca2+ concentration in the terminal. (Middle) FM1-43 intercalates in the membrane and recycles with the exo/endocytic cycle. After washing the excess dye, only the terminals appear labeled. In a last step they can be “destained” to investigate vesicle release kinetics. (Right) Synaptic vesicles of transgenic mice expressing the pH-sensitive probe synaptopHluorin are quenched at rest, because pHluorin is exposed to the inside acidic milieu of the vesicle. Upon vesicle fusion the pHluorin is briefly exposed to the extracellular neutral pH, which can be detected as a subtle increase in fluorescence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Genetically Encoded Markers: synaptopHluorinYet another technique to visualize synaptic activity is based on the genetically engineered fusion protein synaptopHluorin (Figure 3B). Miesenböck et al10Miesenböck G. De Angelis D.A. Rothman J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.Nature. 1998; 394: 192-195Crossref PubMed Scopus (1928) Google Scholar redesigned the naturally occurring green fluorescent protein from the jellyfish Aequorea victoria, to enhance its pH sensitivity in a way that it becomes invisible in an acidic milieu. This pHluorin was then fused to synaptobrevin, a protein present in the synaptic vesicle membrane and important for vesicle fusion at the presynaptic membrane. The inside of synaptic vesicles is acidic and quenches the fluorescence of the pHluorin at rest; upon fusion of the presynaptic vesicle, pHluorin is exposed to the extracellular medium and unquenched, which results in a marked increase in fluorescence. Li et al11Li Z. Burrone J. Tyler W.J. et al.Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.Proc Natl Acad Sci U S A. 2005; 102: 6131-6136Crossref PubMed Scopus (127) Google Scholar were able to make a transgenic synaptopHluorin mouse that expresses the fluorescent marker at the presynaptic sites of many different neurons. In preliminary experiments, we found expression of synaptopHluorin in the enteric nervous system of these transgenic mice (generous gift from V. Murthy, Harvard Medical School). Electrical stimulation, depolarization using high extracellular K+ concentrations, or spontaneous release events lead to a transient increase in fluorescence at the boutons (Figure 2C). Using NH4Cl, which alkalinizes all intracellular compartments, the fluorescence can be unquenched to reveal the structure of synaptic contacts in the enteric ganglia.Dual Color Imaging to Investigate Synaptic ModulationTo investigate which factors modulate synaptic function, dyes with a different spectrum can be used simultaneously. This can be useful to investigate both pre- (red shifted FM595) and postsynaptic signals (Fluo-4) and to address interesting questions on how synaptic function is modulated from “the outside” (circulating or paracrine mediators, pharmacologic agents, adjacent cells) or from “within” the release site (mitochondria). As mentioned, subtle tuning of synaptic communication could drastically alter the output of the ENS. In the central nervous system, (an)orexigenic signaling molecules are able to influence synaptic organization and synaptic signaling,12Diano S. Farr S.A. Benoit S.C. et al.Ghrelin controls hippocampal spine synapse density and memory performance.Nat Neurosci. 2006; 9: 381-388Crossref PubMed Scopus (677) Google Scholar and this has yet to be explored in the ENS.Another interesting topic concerns the role of adjacent and supporting cells. There is strong evidence for the involvement of glial cells in synaptic organization, plasticity, and signaling.13Todd K.J. Serrano A. Lacaille J.C. et al.Glial cells in synaptic plasticity.J Physiol Paris. 2006; 99: 75-83Crossref PubMed Scopus (59) Google Scholar Glial cells are present in enteric ganglia and often outnumber neurons several times. In recent years, it became clear that they are actively involved in controlling gastrointestinal functions. They contain neurotransmitter precursors, have machinery for uptake and degradation of neuroligands, and express neurotransmitter-receptors,14Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (112) Google Scholar which make them well-suited as intermediates in enteric neurotransmission and information processing in the ENS. Third, synaptic transmission requires large amounts of energy and diffusion of adenosine triphosphate (ATP) can be rate-limiting in areas where ATP is consumed rapidly. Mitochondria are vital organelles that, besides producing ATP, also play a crucial role in regulating intracellular Ca2+ concentration.15Vanden Berghe P. Kenyon J.L. et al.Mitochondrial Ca2+ uptake regulates the excitability of myenteric neurons.J Neurosci. 2002; 22: 6962-6971PubMed Google Scholar Translocation and positioning of mitochondria at the presynaptic site is prerequisite for the synapse to optimally function, as seen from the dramatic change in Ca2+ signaling when mitochondria are poisoned. Mitochondrial damage in the ENS is not that extraordinary; because of its location in the gut wall, the ENS is prone to insults, such as inflammation, which can pose substantial oxidative stress on mitochondria present in nerves and nerve endings. Using dual color measurements mitochondrial transport (Mitotracker red) could be monitored simultaneously with Ca2+ events (Fluo-4) in fibers and release sites, which is essential to understand their relationship in normal and diseased conditions. Synaptic imaging is still in its infancy and needs to be improved by further increasing the temporal resolution and refining the techniques to spectrally split different color channels. Light microscopic techniques date back to the end of the 17th century when Anthonie van Leeuwenhoek built an optical device with a series of self-ground lenses. These techniques were instrumental in discovering and elucidating the cellular organization and structure of many organisms. It took until the last decades, to develop fluorescent probes with specific biological or chemical function, before physiologic processes could be monitored. To this end, molecules were designed to either become membrane potential or ion (Ca2+, H+, …) sensitive, or to divulge the location of specific proteins. Apart from the synthetic molecules, genetically encoded protein dyes derived from jellyfish and corals have become extremely useful for labeling specific biological interactions or processes. The use of optical techniques to study cellular function has 2 important advantages. First, several cellular and subcellular structures can be studied simultaneously, which allows accurate detection of sequential events. Second, they always provide a spatial correlate to physiologic events of interest. The enteric nervous system (ENS), responsible for the coordination of many different gastrointestinal functions, consists of approximately 100 million neurons wired in a ganglionated network. Each of the neurons receives synaptic input from many different contacts (Figure 1), which adds layers of complexity to the network. The output of this nerve network controls motility by reflex activation of specific pathways leading to peristalsis. Alternatively, the ENS can be seen as a continuously oscillating network1Wood J.D. Enteric nervous system: reflexes, pattern generators and motility.Curr Opin Gastroenterol. 2008; 24: 149-158Crossref PubMed Scopus (79) Google Scholar that is tilted toward a different output, depending on subtle inputs from the periphery. It is well known that neural networks2Johnson B.R. Schneider L.R. Nadim F. et al.Dopamine modulation of phasing of activity in a rhythmic motor network: contribution of synaptic and intrinsic modulatory actions.J Neurophysiol. 2005; 94: 3101-3111Crossref PubMed Scopus (27) Google Scholar change their output depending on low concentrations of specific mediators. The unitary components that are modulated to produce the change may well be synapses and not neurons. In states of disease, such as inflammatory conditions, synaptic activity was shown to be altered dramatically and persistently.3Linden D.R. Sharkey K.A. Mawe G.M. Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon.J Physiol. 2003; 547: 589-601Crossref PubMed Scopus (162) Google Scholar, 4Lomax A.E. Mawe G.M. Sharkey K.A. Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.J Physiol. 2005; 564: 863-875Crossref PubMed Scopus (78) Google Scholar Therefore, to understand the output of the ENS, it is crucial to also appreciate the contributing synaptic signals, because these may be essential to determine output, although they do not always reach the “attention” level by generating an action potential in the postsynaptic cell. Electrode techniques5Wood J.D. Cellular neurophysiology of enteric neurons.in: Johnson L.R. Barret K.E. Ghisan F.K. Physiology of the gastrointestinal tract. 4th ed. Elsevier, San Diego2006: 629-664Crossref Scopus (6) Google Scholar and voltage-sensitive optical techniques6Schemann M. Michel K. Peters S. et al.Cutting-edge technology III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G919-G925Crossref PubMed Scopus (50) Google Scholar have generated invaluable information with high temporal resolution about fast and slow postsynaptic potentials in the ENS. In this review, we focus on how synaptic activity or neurotransmitter release itself, rather than the processed postsynaptic signal, can be measured optically. Imaging Synaptic EventsCytosolic Ca2+ MeasurementsCytosolic Ca2+ indicators are sensitive to changes in intracellular Ca2+ concentration, which reflect activity of the cell. They can be loaded (1–10 μmol/l, 20 minutes) in their acetoxymethyl (AM)-ester form, which renders the molecule lipophilic and allows it to leak through the cellular membrane. The dye becomes trapped inside the cell once cytosolic esterases have cleaved the AM-ester (Figure 2A). The quality of Ca2+ indicators (Fura-2, Fluo-3, Fluo-4) has improved immensely over the last decades and the signal to noise ratio of dyes like Fluo-4 makes it possible to easily measure signals from small cellular compartments, such as varicosities and synapses. Using this technique, spontaneous and evoked Ca2+ signaling events in fibers and varicosities can be recorded in cultured enteric neurons or in tissue. Using a custom developed computer algorithm (in Igor Pro, Wavemetrics, Lake Oswego, OR) that selects active pixels in an image sequence, we can construct so-called activity over time images that have the same quality of structural information as immunostaining, but in which a physiologic response is represented. Postprocessing of the sample with specific antibodies can further identify the characteristics of the fiber or the apposed postsynaptic cell. Fluo-4 signals have the advantage to be very bright, with a high signal to noise ratio; the loading procedure is in many ways random, but it is difficult, if not impossible, to load synaptic contacts embedded deeply in the tissue. Ca2+ imaging is easy to use and generates high-resolution information about release sites in the ENS, but one should bear in mind that a Fluo-4 signal just reflects the increase in cytosolic Ca2+ and is not conclusive proof for synaptic activity.Labeling Vesicle Recycling: FM1-43Because they were originally meant to become voltage-sensitive probes, FM dyes (FM1-43, FM2-10, FM-595) were designed to intercalate well and fluoresce mainly in a membrane lipid double layer. Because of this property, they became excellent tools to label membrane trafficking and vesicle turnover. The technique, pioneered by Betz and Bewick,7Betz W.J. Bewick G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.Science. 1992; 255: 200-203Crossref PubMed Scopus (648) Google Scholar has generated important information of how synapses organize their synaptic vesicles during neurotransmitter release. The principle is rather simple and involves 3 consecutive steps (Figure 3B). In a first step, FM dye is present in the extracellular medium and actively recycling release sites become labeled, because freshly endocytosed synaptic vesicles at the presynaptic membrane contain the dye. During a second step, the excess dye is washed away from the preparation and the active release sites remain fluorescent (Figure 2B). In a last step, boutons can be stimulated again so that they “destain,” the kinetics of which closely reflect the kinetics of vesicle release (Figure 2B).8Vanden Berghe P. Klingauf J. Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system.Neuroscience. 2007; 145: 88-99Crossref PubMed Scopus (18) Google Scholar With this dye, one can directly measure the dynamic properties of vesicle recycling and release in enteric neurons in primary culture as well as in dissected whole-mount tissue. Using this technique, we were able to show that enteric release sites have kinetic properties very similar to the central nervous system and that brain-derived neurotrophic factor was able to modulate release of enteric nerves caused by subtle electrical stimuli.9Boesmans W. Gomes P. Janssens J. et al.Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system.Gut. 2008; 57: 314-322Crossref PubMed Scopus (64) Google Scholar Owing to the specific loading procedure, FM dyes outperform Ca2+ dyes in selectivity to label synaptic contacts. However, stability of the FM dyes is worse and photobleaching is an important issue the user should consider. A decrease in fluorescence may compromise the “destain” kinetics, bleaching should be reduced as much as possible by keeping exposure times short.Figure 3Schematic of the principles and methods for visualizing synaptic vesicle release. (A) Simplified drawing showing the main characteristics of a presynaptic terminal. Upon arrival of an action potential, voltage gated Ca2+ channels open owing to depolarization and allow Ca2+ to flow into the terminal. This triggers the synaptic vesicles to fuse with the presynaptic membrane and release their neurotransmitter contents. (B) Schematic of 3 different optical techniques to visualize synaptic activity. (Left) The Ca2+ indicator Fluo-4 can be used to monitor the increase in intracellular Ca2+ concentration in the terminal. (Middle) FM1-43 intercalates in the membrane and recycles with the exo/endocytic cycle. After washing the excess dye, only the terminals appear labeled. In a last step they can be “destained” to investigate vesicle release kinetics. (Right) Synaptic vesicles of transgenic mice expressing the pH-sensitive probe synaptopHluorin are quenched at rest, because pHluorin is exposed to the inside acidic milieu of the vesicle. Upon vesicle fusion the pHluorin is briefly exposed to the extracellular neutral pH, which can be detected as a subtle increase in fluorescence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Genetically Encoded Markers: synaptopHluorinYet another technique to visualize synaptic activity is based on the genetically engineered fusion protein synaptopHluorin (Figure 3B). Miesenböck et al10Miesenböck G. De Angelis D.A. Rothman J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.Nature. 1998; 394: 192-195Crossref PubMed Scopus (1928) Google Scholar redesigned the naturally occurring green fluorescent protein from the jellyfish Aequorea victoria, to enhance its pH sensitivity in a way that it becomes invisible in an acidic milieu. This pHluorin was then fused to synaptobrevin, a protein present in the synaptic vesicle membrane and important for vesicle fusion at the presynaptic membrane. The inside of synaptic vesicles is acidic and quenches the fluorescence of the pHluorin at rest; upon fusion of the presynaptic vesicle, pHluorin is exposed to the extracellular medium and unquenched, which results in a marked increase in fluorescence. Li et al11Li Z. Burrone J. Tyler W.J. et al.Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.Proc Natl Acad Sci U S A. 2005; 102: 6131-6136Crossref PubMed Scopus (127) Google Scholar were able to make a transgenic synaptopHluorin mouse that expresses the fluorescent marker at the presynaptic sites of many different neurons. In preliminary experiments, we found expression of synaptopHluorin in the enteric nervous system of these transgenic mice (generous gift from V. Murthy, Harvard Medical School). Electrical stimulation, depolarization using high extracellular K+ concentrations, or spontaneous release events lead to a transient increase in fluorescence at the boutons (Figure 2C). Using NH4Cl, which alkalinizes all intracellular compartments, the fluorescence can be unquenched to reveal the structure of synaptic contacts in the enteric ganglia. Cytosolic Ca2+ MeasurementsCytosolic Ca2+ indicators are sensitive to changes in intracellular Ca2+ concentration, which reflect activity of the cell. They can be loaded (1–10 μmol/l, 20 minutes) in their acetoxymethyl (AM)-ester form, which renders the molecule lipophilic and allows it to leak through the cellular membrane. The dye becomes trapped inside the cell once cytosolic esterases have cleaved the AM-ester (Figure 2A). The quality of Ca2+ indicators (Fura-2, Fluo-3, Fluo-4) has improved immensely over the last decades and the signal to noise ratio of dyes like Fluo-4 makes it possible to easily measure signals from small cellular compartments, such as varicosities and synapses. Using this technique, spontaneous and evoked Ca2+ signaling events in fibers and varicosities can be recorded in cultured enteric neurons or in tissue. Using a custom developed computer algorithm (in Igor Pro, Wavemetrics, Lake Oswego, OR) that selects active pixels in an image sequence, we can construct so-called activity over time images that have the same quality of structural information as immunostaining, but in which a physiologic response is represented. Postprocessing of the sample with specific antibodies can further identify the characteristics of the fiber or the apposed postsynaptic cell. Fluo-4 signals have the advantage to be very bright, with a high signal to noise ratio; the loading procedure is in many ways random, but it is difficult, if not impossible, to load synaptic contacts embedded deeply in the tissue. Ca2+ imaging is easy to use and generates high-resolution information about release sites in the ENS, but one should bear in mind that a Fluo-4 signal just reflects the increase in cytosolic Ca2+ and is not conclusive proof for synaptic activity. Cytosolic Ca2+ indicators are sensitive to changes in intracellular Ca2+ concentration, which reflect activity of the cell. They can be loaded (1–10 μmol/l, 20 minutes) in their acetoxymethyl (AM)-ester form, which renders the molecule lipophilic and allows it to leak through the cellular membrane. The dye becomes trapped inside the cell once cytosolic esterases have cleaved the AM-ester (Figure 2A). The quality of Ca2+ indicators (Fura-2, Fluo-3, Fluo-4) has improved immensely over the last decades and the signal to noise ratio of dyes like Fluo-4 makes it possible to easily measure signals from small cellular compartments, such as varicosities and synapses. Using this technique, spontaneous and evoked Ca2+ signaling events in fibers and varicosities can be recorded in cultured enteric neurons or in tissue. Using a custom developed computer algorithm (in Igor Pro, Wavemetrics, Lake Oswego, OR) that selects active pixels in an image sequence, we can construct so-called activity over time images that have the same quality of structural information as immunostaining, but in which a physiologic response is represented. Postprocessing of the sample with specific antibodies can further identify the characteristics of the fiber or the apposed postsynaptic cell. Fluo-4 signals have the advantage to be very bright, with a high signal to noise ratio; the loading procedure is in many ways random, but it is difficult, if not impossible, to load synaptic contacts embedded deeply in the tissue. Ca2+ imaging is easy to use and generates high-resolution information about release sites in the ENS, but one should bear in mind that a Fluo-4 signal just reflects the increase in cytosolic Ca2+ and is not conclusive proof for synaptic activity. Labeling Vesicle Recycling: FM1-43Because they were originally meant to become voltage-sensitive probes, FM dyes (FM1-43, FM2-10, FM-595) were designed to intercalate well and fluoresce mainly in a membrane lipid double layer. Because of this property, they became excellent tools to label membrane trafficking and vesicle turnover. The technique, pioneered by Betz and Bewick,7Betz W.J. Bewick G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.Science. 1992; 255: 200-203Crossref PubMed Scopus (648) Google Scholar has generated important information of how synapses organize their synaptic vesicles during neurotransmitter release. The principle is rather simple and involves 3 consecutive steps (Figure 3B). In a first step, FM dye is present in the extracellular medium and actively recycling release sites become labeled, because freshly endocytosed synaptic vesicles at the presynaptic membrane contain the dye. During a second step, the excess dye is washed away from the preparation and the active release sites remain fluorescent (Figure 2B). In a last step, boutons can be stimulated again so that they “destain,” the kinetics of which closely reflect the kinetics of vesicle release (Figure 2B).8Vanden Berghe P. Klingauf J. Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system.Neuroscience. 2007; 145: 88-99Crossref PubMed Scopus (18) Google Scholar With this dye, one can directly measure the dynamic properties of vesicle recycling and release in enteric neurons in primary culture as well as in dissected whole-mount tissue. Using this technique, we were able to show that enteric release sites have kinetic properties very similar to the central nervous system and that brain-derived neurotrophic factor was able to modulate release of enteric nerves caused by subtle electrical stimuli.9Boesmans W. Gomes P. Janssens J. et al.Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system.Gut. 2008; 57: 314-322Crossref PubMed Scopus (64) Google Scholar Owing to the specific loading procedure, FM dyes outperform Ca2+ dyes in selectivity to label synaptic contacts. However, stability of the FM dyes is worse and photobleaching is an important issue the user should consider. A decrease in fluorescence may compromise the “destain” kinetics, bleaching should be reduced as much as possible by keeping exposure times short. Because they were originally meant to become voltage-sensitive probes, FM dyes (FM1-43, FM2-10, FM-595) were designed to intercalate well and fluoresce mainly in a membrane lipid double layer. Because of this property, they became excellent tools to label membrane trafficking and vesicle turnover. The technique, pioneered by Betz and Bewick,7Betz W.J. Bewick G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.Science. 1992; 255: 200-203Crossref PubMed Scopus (648) Google Scholar has generated important information of how synapses organize their synaptic vesicles during neurotransmitter release. The principle is rather simple and involves 3 consecutive steps (Figure 3B). In a first step, FM dye is present in the extracellular medium and actively recycling release sites become labeled, because freshly endocytosed synaptic vesicles at the presynaptic membrane contain the dye. During a second step, the excess dye is washed away from the preparation and the active release sites remain fluorescent (Figure 2B). In a last step, boutons can be stimulated again so that they “destain,” the kinetics of which closely reflect the kinetics of vesicle release (Figure 2B).8Vanden Berghe P. Klingauf J. Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system.Neuroscience. 2007; 145: 88-99Crossref PubMed Scopus (18) Google Scholar With this dye, one can directly measure the dynamic properties of vesicle recycling and release in enteric neurons in primary culture as well as in dissected whole-mount tissue. Using this technique, we were able to show that enteric release sites have kinetic properties very similar to the central nervous system and that brain-derived neurotrophic factor was able to modulate release of enteric nerves caused by subtle electrical stimuli.9Boesmans W. Gomes P. Janssens J. et al.Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system.Gut. 2008; 57: 314-322Crossref PubMed Scopus (64) Google Scholar Owing to the specific loading procedure, FM dyes outperform Ca2+ dyes in selectivity to label synaptic contacts. However, stability of the FM dyes is worse and photobleaching is an important issue the user should consider. A decrease in fluorescence may compromise the “destain” kinetics, bleaching should be reduced as much as possible by keeping exposure times short. Genetically Encoded Markers: synaptopHluorinYet another technique to visualize synaptic activity is based on the genetically engineered fusion protein synaptopHluorin (Figure 3B). Miesenböck et al10Miesenböck G. De Angelis D.A. Rothman J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.Nature. 1998; 394: 192-195Crossref PubMed Scopus (1928) Google Scholar redesigned the naturally occurring green fluorescent protein from the jellyfish Aequorea victoria, to enhance its pH sensitivity in a way that it becomes invisible in an acidic milieu. This pHluorin was then fused to synaptobrevin, a protein present in the synaptic vesicle membrane and important for vesicle fusion at the presynaptic membrane. The inside of synaptic vesicles is acidic and quenches the fluorescence of the pHluorin at rest; upon fusion of the presynaptic vesicle, pHluorin is exposed to the extracellular medium and unquenched, which results in a marked increase in fluorescence. Li et al11Li Z. Burrone J. Tyler W.J. et al.Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.Proc Natl Acad Sci U S A. 2005; 102: 6131-6136Crossref PubMed Scopus (127) Google Scholar were able to make a transgenic synaptopHluorin mouse that expresses the fluorescent marker at the presynaptic sites of many different neurons. In preliminary experiments, we found expression of synaptopHluorin in the enteric nervous system of these transgenic mice (generous gift from V. Murthy, Harvard Medical School). Electrical stimulation, depolarization using high extracellular K+ concentrations, or spontaneous release events lead to a transient increase in fluorescence at the boutons (Figure 2C). Using NH4Cl, which alkalinizes all intracellular compartments, the fluorescence can be unquenched to reveal the structure of synaptic contacts in the enteric ganglia. Yet another technique to visualize synaptic activity is based on the genetically engineered fusion protein synaptopHluorin (Figure 3B). Miesenböck et al10Miesenböck G. De Angelis D.A. Rothman J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.Nature. 1998; 394: 192-195Crossref PubMed Scopus (1928) Google Scholar redesigned the naturally occurring green fluorescent protein from the jellyfish Aequorea victoria, to enhance its pH sensitivity in a way that it becomes invisible in an acidic milieu. This pHluorin was then fused to synaptobrevin, a protein present in the synaptic vesicle membrane and important for vesicle fusion at the presynaptic membrane. The inside of synaptic vesicles is acidic and quenches the fluorescence of the pHluorin at rest; upon fusion of the presynaptic vesicle, pHluorin is exposed to the extracellular medium and unquenched, which results in a marked increase in fluorescence. Li et al11Li Z. Burrone J. Tyler W.J. et al.Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.Proc Natl Acad Sci U S A. 2005; 102: 6131-6136Crossref PubMed Scopus (127) Google Scholar were able to make a transgenic synaptopHluorin mouse that expresses the fluorescent marker at the presynaptic sites of many different neurons. In preliminary experiments, we found expression of synaptopHluorin in the enteric nervous system of these transgenic mice (generous gift from V. Murthy, Harvard Medical School). Electrical stimulation, depolarization using high extracellular K+ concentrations, or spontaneous release events lead to a transient increase in fluorescence at the boutons (Figure 2C). Using NH4Cl, which alkalinizes all intracellular compartments, the fluorescence can be unquenched to reveal the structure of synaptic contacts in the enteric ganglia. Dual Color Imaging to Investigate Synaptic ModulationTo investigate which factors modulate synaptic function, dyes with a different spectrum can be used simultaneously. This can be useful to investigate both pre- (red shifted FM595) and postsynaptic signals (Fluo-4) and to address interesting questions on how synaptic function is modulated from “the outside” (circulating or paracrine mediators, pharmacologic agents, adjacent cells) or from “within” the release site (mitochondria). As mentioned, subtle tuning of synaptic communication could drastically alter the output of the ENS. In the central nervous system, (an)orexigenic signaling molecules are able to influence synaptic organization and synaptic signaling,12Diano S. Farr S.A. Benoit S.C. et al.Ghrelin controls hippocampal spine synapse density and memory performance.Nat Neurosci. 2006; 9: 381-388Crossref PubMed Scopus (677) Google Scholar and this has yet to be explored in the ENS.Another interesting topic concerns the role of adjacent and supporting cells. There is strong evidence for the involvement of glial cells in synaptic organization, plasticity, and signaling.13Todd K.J. Serrano A. Lacaille J.C. et al.Glial cells in synaptic plasticity.J Physiol Paris. 2006; 99: 75-83Crossref PubMed Scopus (59) Google Scholar Glial cells are present in enteric ganglia and often outnumber neurons several times. In recent years, it became clear that they are actively involved in controlling gastrointestinal functions. They contain neurotransmitter precursors, have machinery for uptake and degradation of neuroligands, and express neurotransmitter-receptors,14Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (112) Google Scholar which make them well-suited as intermediates in enteric neurotransmission and information processing in the ENS. Third, synaptic transmission requires large amounts of energy and diffusion of adenosine triphosphate (ATP) can be rate-limiting in areas where ATP is consumed rapidly. Mitochondria are vital organelles that, besides producing ATP, also play a crucial role in regulating intracellular Ca2+ concentration.15Vanden Berghe P. Kenyon J.L. et al.Mitochondrial Ca2+ uptake regulates the excitability of myenteric neurons.J Neurosci. 2002; 22: 6962-6971PubMed Google Scholar Translocation and positioning of mitochondria at the presynaptic site is prerequisite for the synapse to optimally function, as seen from the dramatic change in Ca2+ signaling when mitochondria are poisoned. Mitochondrial damage in the ENS is not that extraordinary; because of its location in the gut wall, the ENS is prone to insults, such as inflammation, which can pose substantial oxidative stress on mitochondria present in nerves and nerve endings. Using dual color measurements mitochondrial transport (Mitotracker red) could be monitored simultaneously with Ca2+ events (Fluo-4) in fibers and release sites, which is essential to understand their relationship in normal and diseased conditions. Synaptic imaging is still in its infancy and needs to be improved by further increasing the temporal resolution and refining the techniques to spectrally split different color channels. To investigate which factors modulate synaptic function, dyes with a different spectrum can be used simultaneously. This can be useful to investigate both pre- (red shifted FM595) and postsynaptic signals (Fluo-4) and to address interesting questions on how synaptic function is modulated from “the outside” (circulating or paracrine mediators, pharmacologic agents, adjacent cells) or from “within” the release site (mitochondria). As mentioned, subtle tuning of synaptic communication could drastically alter the output of the ENS. In the central nervous system, (an)orexigenic signaling molecules are able to influence synaptic organization and synaptic signaling,12Diano S. Farr S.A. Benoit S.C. et al.Ghrelin controls hippocampal spine synapse density and memory performance.Nat Neurosci. 2006; 9: 381-388Crossref PubMed Scopus (677) Google Scholar and this has yet to be explored in the ENS. Another interesting topic concerns the role of adjacent and supporting cells. There is strong evidence for the involvement of glial cells in synaptic organization, plasticity, and signaling.13Todd K.J. Serrano A. Lacaille J.C. et al.Glial cells in synaptic plasticity.J Physiol Paris. 2006; 99: 75-83Crossref PubMed Scopus (59) Google Scholar Glial cells are present in enteric ganglia and often outnumber neurons several times. In recent years, it became clear that they are actively involved in controlling gastrointestinal functions. They contain neurotransmitter precursors, have machinery for uptake and degradation of neuroligands, and express neurotransmitter-receptors,14Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (112) Google Scholar which make them well-suited as intermediates in enteric neurotransmission and information processing in the ENS. Third, synaptic transmission requires large amounts of energy and diffusion of adenosine triphosphate (ATP) can be rate-limiting in areas where ATP is consumed rapidly. Mitochondria are vital organelles that, besides producing ATP, also play a crucial role in regulating intracellular Ca2+ concentration.15Vanden Berghe P. Kenyon J.L. et al.Mitochondrial Ca2+ uptake regulates the excitability of myenteric neurons.J Neurosci. 2002; 22: 6962-6971PubMed Google Scholar Translocation and positioning of mitochondria at the presynaptic site is prerequisite for the synapse to optimally function, as seen from the dramatic change in Ca2+ signaling when mitochondria are poisoned. Mitochondrial damage in the ENS is not that extraordinary; because of its location in the gut wall, the ENS is prone to insults, such as inflammation, which can pose substantial oxidative stress on mitochondria present in nerves and nerve endings. Using dual color measurements mitochondrial transport (Mitotracker red) could be monitored simultaneously with Ca2+ events (Fluo-4) in fibers and release sites, which is essential to understand their relationship in normal and diseased conditions. Synaptic imaging is still in its infancy and needs to be improved by further increasing the temporal resolution and refining the techniques to spectrally split different color channels.